12‐lipoxygenase isoenzymes in mouse skin tumor development

Abstract

12‐lipoxygenase‐catalyzed arachidonic acid metabolism in normal and neoplastic mouse epidermis was assessed by cDNA cloning of the epidermal 12‐lipoxygenases and by studying their expression patterns, enzyme activities, and product levels. Papillomas and squamous cell carcinomas induced by the initiation/promotion protocol contained 50‐to 60‐fold more 12‐hydroxy‐5,8,10,14‐eicosatetraenoic acid (a) than normal epidermis. The ratio of S to R enantiomers was 9:1. This indicates that most of this eicosanoid was of enzymatic origin. Accordingly, cell‐free preparations of the tumors exhibited about fivefold elevated 12‐lipoxygenase activities. A papilloma‐derived cDNA library was screened with human platelet‐type 12‐lipoxygenase cDNA probes. Two cDNA clones encoding the platelet‐type and the leukocyte‐type isoforms of murine 12‐lipoxygenase were isolated, demonstrating the coexpression of the isoenzymes in the same tissue and species. When expressed in COS‐7 cells, the recombinant enzymes showed the characteristic substrate selectivity and product profile, with the leukocyte‐type enzyme metabolizing linoleic and arachidonic acid to 13‐hydroxy‐9,11‐octadecadienoic acid and to 12‐ and 15‐HETE, respectively, and the platelet‐type enzyme oxygenating exclusively arachidonic acid to 12‐HETE. In epidermis in vivo and in keratinocytes in culture, only the platelet‐type 12‐lipoxygenase (mRNA and protein) was detectable. In mouse epidermis both isoenzymes were induced transiently by phorbol esters. Most tumors showed constitutive overexpression of platelet‐type mRNA, whereas leukocyte‐type specific transcripts were detectable only in a few tumors. These data suggest that the platelet‐type enzyme is the 12‐lipoxygenase isoform of keratinocytes that is responsible for the generation of most of the 12‐HETE found in neoplastic epidermis. ©1995 Wiley‐Liss, Inc.